Unique Carboxy-terminal Domain Research Articles

Molecular mechanism of the RNA helicase DHX37 and its activation by UTP14A in ribosome biogenesis.

Eukaryotic ribosome biogenesis is a highly orchestrated process involving numerous assembly factors including ATP-dependent RNA helicases. The DEAH helicase DHX37 (Dhr1 in yeast) is activated by the ribosome biogenesis factor UTP14A to facilitate maturation of the small ribosomal subunit. We report the crystal structure of DHX37 in complex with single-stranded RNA, revealing a canonical DEAH ATPase/helicase architecture complemented by a structurally unique carboxy-terminal domain (CTD). Structural comparisons of the nucleotide-free DHX37-RNA complex with DEAH helicases bound to RNA and ATP analogs reveal conformational changes resulting in a register shift in the bound RNA, suggesting a mechanism for ATP-dependent 3′–5′ RNA translocation. We further show that a conserved sequence motif in UTP14A interacts with and activates DHX37 by stimulating its ATPase activity and enhancing RNA binding. In turn, the CTD of DHX37 is required, but not sufficient, for interaction with UTP14A in vitro and is essential for ribosome biogenesis in vivo. Together, these results shed light on the mechanism of DHX37 and the function of UTP14A in controlling its recruitment and activity during ribosome biogenesis.

Carboxy-terminal phosphorylation of SIRT1 by protein kinase CK2

Previous analyses of the sirtuin family of histone deacetylases and its most prominent member SIRT1 have focused primarily on the identification of cellular targets exploring the underlying molecular mechanisms of its implicated function in the control of metabolic homeostasis, differentiation, apoptosis and cell survival. So far, little is known about the regulation of SIRT1 itself. In the study presented herein, we assigned the main region of SIRT1 in vivo phosphorylation to amino acids 643–691 of the unique carboxy-terminal domain. Furthermore, we demonstrate that SIRT1 is a substrate for protein kinase CK2 both in vitro and in vivo. Both, deletion construct analyses and serine-to-alanine mutations identified SIRT1 Ser-659 and Ser-661 as major CK2 phosphorylation sites that are phosphorylated in vivo as well.

Identification of full genes and proteins of MCM9, a novel, vertebrate-specific member of the MCM2–8 protein family

MCM2–7 proteins are conserved replication factors functioning as DNA helicases during DNA synthesis. MCM8 is another member of this family, which appears to be specific for higher eukaryotes, as it is absent in worms and yeast. Here we report the complete identification of a novel member of this family, the MCM9 protein. Like MCM8, MCM9 is only present in the genome of higher eukaryotes. This protein contains an MCM8-like ATP binding and hydrolysis motif implicated in helicase activity. Strikingly, MCM9 also contains a unique carboxy-terminal domain which has only weak homology to MCM2–7 and MCM8 but is conserved within MCM9 homologs. We also show that the very recently reported human MCM9 protein (HsMCM9), which resembles a truncated MCM-like protein missing a part of the MCM2–7 signature domain, is an incomplete form of the full length HsMCM9 described here. Searching the human genome with either the newly identified human MCM9 or other MCM protein sequences, we did not detect further additional members of this DNA helicase family and suggest that it is constituted of eight members, falling into two different groups, one constituted by the MCM2–7 complex and the other by MCM8 and MCM9, which are present only in higher eukaryotes.

Actively Transcribing RNA Polymerase II Concentrates on Spliced Leader Genes in the Nucleus of Trypanosoma cruzi

RNA polymerase II of trypanosomes, early diverging eukaryotes, transcribes long polycistronic messages, which are not capped but are processed by trans-splicing and polyadenylation to form mature mRNAs. The same RNA polymerase II also transcribes the genes coding for the spliced leader RNA, which are capped, exported to the cytoplasm, processed, and reimported into the nucleus before they are used as splicing donors to form mRNAs from pre-mRNA polycistronic transcripts. As pre-mRNA and spliced leader transcription events appear to be uncoupled, we studied how the RNA polymerase II is distributed in the nucleus of Trypanosoma cruzi. Using specific antibodies to the T. cruzi RNA polymerase II unique carboxy-terminal domain, we demonstrated that large amounts of the enzyme are found concentrated in a domain close to the parasite nucleolus and containing the spliced leader genes. The remaining RNA polymerase II is diffusely distributed in the nucleoplasm. The spliced leader-associated RNA polymerase II localization is dependent on the cell transcriptional state. It disperses when transcription is blocked by alpha-amanitin and actinomycin D. Tubulin genes are excluded from this domain, suggesting that it may exclusively be the transcriptional site of spliced leader genes. Trypomastigote forms of the parasite, which have reduced spliced leader transcription, show less RNA polymerase II labeling, and the spliced leader genes are more dispersed in the nucleoplasm. These results provide strong evidences that transcription of spliced leader RNAs occurs in a particular domain in the T. cruzi nucleus.

P21Waf1/Cip1 and the prevention of oxidative stress.

pulmonary dysfunction resulting from acute oxygen toxicity is due at least in part to the death of lung epithelial cells. Pulmonary epithelial cells are essential to the maintenance of the alveolar-capillary barrier, and compromise may lead to leakage of fluid and proteins in the airways and alveoli

MST4, a new Ste20-related kinase that mediates cell growth and transformation via modulating ERK pathway.

In this study, we report the cloning and characterization of a novel human Ste20-related kinase that we designated MST4. The 416 amino acid full-length MST4 contains an amino-terminal kinase domain, which is highly homologous to MST3 and SOK, and a unique carboxy-terminal domain. Northern blot analysis indicated that MST4 is highly expressed in placenta, thymus, and peripheral blood leukocytes. Wild-type but not kinase-dead MST4 can phosphorylate myelin basic protein in an in vitro kinase assay. MST4 specifically activates ERK but not JNK or p38 MAPK in transient transfected cells or in stable cell lines. Overexpression of dominant negative MEK1 or treatment with PD98059 abolishes MST4-induced ERK activity, whereas dominant-negative Ras or c-Raf-1 mutants failed to do so, indicating MST4 activates MEK1/ERK via a Ras/Raf-1 independent pathway. HeLa and Phoenix cell lines overexpressing wild-type, but not kinase-dead, MST4 exhibit increased growth rate and form aggressive soft-agar colonies. These phenotypes can be inhibited by PD98059. These results provide the first evidence that MST4 is biologically active in the activation of MEK/ERK pathway and in mediating cell growth and transformation.

AOP2 (antioxidant protein 2): structure and function of a unique thiol-specific antioxidant.

The accumulation of reactive oxygen species (ROS) in response to extracellular signals or intracellular biochemical processes can be regulated by the coordinate action of many antioxidant proteins. Because moderate levels of ROS can act as intracellular messengers in many of these processes, this modulation is critical for the transduction of specific signals. The thiol-specific antioxidant (TSA) family is a highly conserved group of enzymes that can reduce hydroperoxides in the presence of a thiol-containing electron donor. AOP2 (antioxidant protein 2) is a newly described member that shows significant evolutionary conservation between many different organisms. The protein contains three motifs that are highly conserved within the TSA family, including a cysteine residue that is the active site of oxidation for this class of proteins. Although AOP2 possesses TSA activity, it has several unique characteristics, including the absence of a second cysteine residue that is conserved in all other TSA proteins, the presence of a unique carboxy-terminal domain, and a demonstrated phospholipase activity. Furthermore, AOP2 shows conservation of several amino acids important in dimer formation and active site configuration that are not found in the other family members. Together, these data strongly suggest that AOP2 is a novel thiol-dependent antioxidant that functions to scavenge particular hydroperoxides in the cell and mediate specific signals. There is also evidence supporting a role for AOP2 in certain disease processes including atherosclerosis. Further evaluation of this protein and its substrate specificity will likely shed light on its precise role in cellular oxidant defense, signal transduction and pathogenesis.

The Role of the Carboxy Terminus of Tropoelastin in its Assembly into the Elastic Fiber

A tribute to Dr. Ines Mandl by Carl Franzblau This article is dedicated to the 80th birthday of Dr. Ines Mandl, who for many years was a colleague of ours in the elastin world. My immediate recollection of the younger Ines is a bubbly, vibrant scientist full of enthusiasm and ideas. She was quick on her feet and willing to collaborate and cooperate. Her contributions to our understanding of elastases, collagenases and the respective proteins they digest were immense. Our group is always referring to critical publications that she and her colleagues at Columbia University have published. The field of connective tissue has come a long way, and in no small part it is due to the contributions of our colleague, Ines Mandl.Tropoelastin, the soluble precursor protein of insoluble amorphous elastin, contains repeating segments that are important for the characteristic elasticity and crosslinking sites of mature elastin. In addition, there is a unique carboxy terminal domain that is encoded by exon 36 of the elastin gene, and it has been suggested that this region may play a role in the process of insolubilization. The contribution of exon 36 to the maturation of tropoelastin into insoluble elastin was probed in these studies. Neonatal rat aortic smooth muscle cells were cultured and the fate of [3H] Lys labeled human recombinant tropoelastin (hrTE) molecules added to the cultures was monitored. In comparison to the hrTE containing the region encoded by exon 36, hrTE molecules lacking this domain were less efficiently incorporated into elastin, as evidenced by a decrease in NaOH insoluble radioactivity. Specific residues within the domain encoded by exon 36 were targeted for further study in experiments in which the two Cys residues were reduced and alkylated, and/or the four basic Arg-Lys-Arg-Lys residues at the carboxy terminus were removed. Both of these modifications resulted in decreased incorporation into elastin equivalent to the complete removal of the carboxy terminus. Prior treatment of the cell layer with elastase reduced the efficiency of insolubilization of hrTE containing the domain encoded by exon 36, but had no effect on the processing of molecules lacking this region. These data suggest that exon 36 of the elastin gene contributes to normal efficient incorporation of tropoelastin into the elastin fiber.

Interactions between dystrophin glycoprotein complex proteins

The organization of the dystrophin glycoprotein complex (DGC) was studied by investigating interactions between its components. For this purpose, mouse dystrophin and syntrophin-1 (alpha-syntrophin) sequences were expressed as chimeric fusion proteins and used in overlay binding experiments to probe gel blots of purified rabbit muscle DGC. In order to identify the DGC proteins that bind to different regions of dystrophin, the amino-terminal 385 amino acids, the unique carboxy-terminal domain (amino acids 3266-3678), and the adjacent cysteine-rich region of dystrophin homologous to alpha-actinin (amino acids 3074-3265) were expressed as separate fusion proteins. The cysteine-rich sequences of dystrophin predominantly bound adhalin (gp50) and to full length dystrophin suggesting that these sequences may also be important to dystrophin dimerization. The carboxy-terminal domain sequences strongly bound all of the DGC syntrophins and weakly, adhalin, while the amino-terminal sequences of dystrophin bound none of the proteins of this complex. Fusion proteins containing alpha-syntrophin sequences bound not only to dystrophin but also to all three DGC syntrophins, adhalin, and gp35. The interactions identified here were used to refine the existing model of DGC organization to make it consistent with the current data.

Measles Virus V Protein Binds Zinc

Measles virus transcription generates multiple P/C gene-specific mRNAs by a process which has been termed editing. In one of these mRNAs, the cotranscriptional addition of a single nontemplated G residue allows translational access to the V protein reading frame. The protein translated from this mRNA has been called V and consists of 231 amino-terminal amino acid residues identical to those at the amino terminus of the P protein followed by a unique carboxy-terminal domain consisting of 68 amino acids from the V reading frame. The most striking feature of this unique domain is the presence within it of seven cysteine residues whose presence and position are highly conserved among different paramyxoviruses. The number and arrangement of these cysteine residues is suggestive of a zinc finger protein. We have used a zinc binding protocol to determine that V protein does indeed bind zinc, have further demonstrated that this metal binding activity is highly specific to zinc, and have shown that it is the unique carboxy-terminal domain of the V protein that is responsible for zinc binding.

Redistribution of nuclear lamin A is an early event associated with differentiation of human promyelocytic leukemia HL-60 cells.

The nuclear lamina of mammalian somatic cells is characterized by the constitutive presence of lamin B polypeptides while the appearance of lamins A and C generally occur during establishment of a differentiated phenotype. We have used antibodies specific to the unique carboxy-terminal domain of lamin A, i.e. distinct from the shared domains of lamins A and C, to study the individual behaviour of lamin A during establishment of a macrophage phenotype in human HL-60 cells. Lamin A was present as a nuclear cap in the majority of undifferentiated cells and it was redistributed to a full peripheral nuclear location very early after induction of differentiation by phorbol esters, even in the presence of a protein synthesis inhibitor. Induction of the cells into a reversible precommitment state by bromodeoxyuridine was accompanied by a similar redistribution of lamin A that however reverted to a cap after removal of inducer. No changes were observed in the uniform peripheral nuclear location of lamin C under all of these conditions. These results strongly suggest that lamin A plays a role in the early events of cell differentiation. Taken together with previous results on the interaction of A-type lamins with chromatin, these findings offer experimental evidence consistent with the proposed role of A-type lamins, and particularly lamin A, in the process of chromatin reorganization that accompanies the expression of a differentiated phenotype.

RNA polymerase II carboxy-terminal domain contributes to the response to multiple acidic activators in vitro.

The largest subunit of RNA polymerase II contains a unique carboxy-terminal domain (CTD) that consists of repeats of the heptapeptide YSPTSPS. RNA polymerase II CTD truncation mutations affect the ability to induce transcription of a subset of yeast genes in vivo, and the lack of response to induction maps to the upstream activating sequences of these genes. Here, we report that progressive truncation of the yeast RNA polymerase II CTD causes progressive loss of trans-activator-dependent transcription in nuclear extracts but has little effect on elongation or termination. Specific transcription, which is reduced by up to 50-fold in these assays, can be restored in the defective nuclear extracts by adding purified wild-type RNA polymerase II. The defects in factor-dependent transcription are observed with templates that are assembled into nucleosomes as well as with templates that are not so assembled. Defects in factor-independent transcription are also observed, but these are not as profound as those observed in the presence of trans-activators. These results indicate that the RNA polymerase II CTD functions during transcription initiation and is required for normal levels of activated transcription in vitro.